Ab16667

The tumor tissues were fixed in 10% neutral-buffered paraformaldehyde, followed by immersion in liquid paraffin, and sectioned (5-μm thickness). Then, the samples were stained with hematoxylin and eosin and with antibodies against Ki-67 (Abcam, ab16667), NF-κB p65 (Santa Cruz, sc514451), p-IκBα (Santa Cruz, sc8404), p21 (Proteintech, #10355-1-AP), CDK4 (Epitomics, #3830-1), CDK6 (Proteintech, #14052-1-AP), CDK2 (Abcam ab32147), p-Rb (Ser807, Abcam, ab184796), E2F1 (St John’s Laboratory, STJ92807), Skp2 (Santa Cruz, sc7164), c-MYC (Abcam, ab32072), and p27 (sc528, Santa Cruz). Finally, the sections were mounted with DPX Mountant (Sigma, 317616) for histological analysis. Staining scores were noted by the intensity and percentage of positively stained cells. The percentage of positive tumor cells was divided into four grades: 0 (<5% positive), 1 (<25% positive), 2 (25–50% positive), 3 for (51–75% positive), and 4 (>75% positive). The intensity of immunostaining was scored as follows: 0 (no staining), 1 (weak staining), 2 (intermediate staining), or 3 (strong staining). Ten random fields were selected and viewed at ×400 in each section to obtain an average score (Li et al., 2020 (link)).

Muscle sections attached to positively charged frosted glass microscope slides were fixed with 70% ethanol at 4 °C overnight and rinsed with 1× phosphate-buffered saline (PBS) the next day. The sections were then blocked for 30 min in 1% staining buffer (1% calf serum in 1× PBS). Samples were permeabilized with 0.1% Triton X-100 in 1X PBS for 5 min at room temperature and subsequently rinsed with 1% staining buffer 3 times, 5 min per rinse. The sections were incubated with primary antibodies diluted to 0.5–1 μg/ml at 4 °C overnight. The slides were rinsed as stated and then coated with secondary antibodies and Hoechst nuclear DNA stain for 2 h at room temperature in the dark. Samples were rinsed with PBS and mounted with Fluoromount. A Zeiss Axioscope fluorescent microscope was used for imaging. antibodies: rabbit anti-GFP antibody (abcam290,1:5000), rabbit anti-Ki67(ab16667, 1:200), and mouse anti-dystrophin antibody (Santa Cruz Biotechnology 58754,1:200) were used at dilutions according to manufacturer’s instructions. Secondary fluorochrome conjugated antibodies were from Life Technologies: goat anti-rabbit 546 (A11010, 1:2000) and goat anti-mouse 488 (A11029, 1:2000). DNA was stained by Hoechst 33342 from Sigma Aldrich (B2261) 1:500 at 1 μg/mL.